In addition to sequence-dependent changes to the DNA, tumour cells also differ on account of sequence-independent epigenetic DNA alterations, including hypermethylation. These tumour-specific changes in the DNA methylation can be used as new cancer markers (e.g. WO2012007462A1, WO2013064163A1, US20130022974, US20110301050).
However, these altered methylation patterns in selective DNA portions can currently only be detected in blood, urine or other human bodily fluids or smears and histological preparations to an insufficient level of analytical sensitivity. Currently, the methylation level in various targets of interest is mainly determined using PCR-based methods, which are carried out following bisulphite pre-treatment of genomic DNA, apart from in the case of methylation-sensitive restriction enzyme analysis (MSRE-PCR). The detection limits differ between the various methods for detecting methylated DNA. Direct BSP (Sanger sequencing) has a sensitivity of from 10-20%, whereas pyrosequencing and MALDI-TOF mass spectrometry-based methods achieve sensitivities of around 5% [1, 2]. MSP (methylation specific PCR), MethyLight, SMART-MSP (sensitive melting analysis after real time-methylation specific PCR) and MS-HRM (methylation-sensitive high-resolution melting) have a detection sensitivity of between 0.1-1.0% [3-6]. PCR bias is discussed as being a significant drawback of current PCR-based methods; this is a phenomenon whereby methylated and unmethylated DNA strands are copied at different levels of efficiency [3, 4].
Another problem associated with current DNA methylation detection methods is that of false positives, which may occur due to incomplete bisulphite conversion and non-specific primer annealing, in particular when methyl-specific primers are used [4]. In addition, none of the aforementioned methods involves a sensitive and above all quantitative detection of heterogeneously methylated DNA fragments, known as “epialleles”. Digital PCR is a new technique in which no PCR bias occurs since each DNA molecule is copied in a separate reaction compartment [7, 8]. Regardless of the amount of DNA available and the assay design, sensitivities of up to 0.001% are described for dPCR [9]. Another advantage of this technique is that absolute values are obtained even when no calibrators are used.
Previously, a commercial test based on detecting cellular epigenetic changes such as DNA methylation as an early and typical feature of malignant changes was only available for diagnosing colorectal cancer by identifying the methylated SEPT9 [10], diagnosing gliomas by means of MGMT, and diagnosing lung cancer by detecting SHOX2 [11]. The SEPT9 test (Epigenomics AG, Berlin, Del.) has a diagnostic sensitivity of 72% and a diagnostic specificity of 90% for detecting colon cancer, i.e. 10% of the individuals tested are given pathological results despite there being no colon cancer [10]. For the GSTP1 gene, the LightMix Kit GSTP1 (Epigenomics, Berlin, Del.) is commercially available, although insufficiently high analytical sensitivity has meant this test has yet to be widely used in conjunction with PSA determination in the clinical-chemical diagnosis of prostate cancer. The ConfirmMDx test marketed by MDxHealth for detecting or ruling out prostate cancer in prostate tissue biopsy samples has a diagnostic sensitivity of 68% and a diagnostic specificity of 64% for detecting prostate tumour cells in the tissues tested, i.e. for 36% of the tissue samples tested, pathological findings are issued despite there being no prostate cancer [12].
The importance of establishing new biomarkers and providing corresponding commercial test kits, e.g. for diagnosing prostate cancer (PCa) can be seen in the high number (more than 150,000) of prostate biopsies carried out annually in Germany alone that were indicated on the basis of PSA determinations and in which tumours were only detected in around 25% of the cases studied [13, 14]. Prostate needle biopsies are an invasive diagnostic method associated with side effects requiring treatment such as bleeds and inflammation in around 5% of biopsies [13, 14]. In addition, not all PCa are detected with a single tissue biopsy, meaning that repeat tests are necessary; a negative result does not reliably rule out the possibility of PCa being present either [13, 14].
The object of the invention is to specify an improved method for diagnosing tumours and a kit for carrying out said method that are suitable in particular for early diagnosis (screening) and for distinguishing between benign and malignant tumours by means of PCR, in particular in bodily fluids.
The object is achieved by a method for diagnosing a tumour disease in an isolated sample, comprising the steps of:    a) bisulphite conversion of the DNA in the sample (conversion of unmethylated cytosine into uracil),    b) (pre-)amplification of methylated and unmethylated DNA sequences by means of PCR, the methylated DNA sequences (where these are tumour-specific) preferably being amplified to a greater extent than the unmethylated DNA sequences or alternatively the unmethylated DNA sequences (where these are tumour-specific) being amplified over the methylated DNA sequences, and then    c) quantifying the pre-amplified methylated and unmethylated DNA by means of digital PCR (dPCR), the number of pre-amplified DNA copies used in the dPCR preferably being above the normal Poisson distribution.
Step b) includes pre-amplifying genomic DNA by means of optimised bias-based PCR, the bias preferably being in favour of the methylated sequences or alternatively in favour of the unmethylated sequences (depending on the specificity for tumour DNA). Therefore, this step will also be referred to as bias-based PCR amplification (BBPA) below. In this step, genomic DNA sequences (referred to in the following as targets of interest or target sequences) that are known to be methylated in malignant tumours are each amplified by means of specific primer pairs such that corresponding unmethylated sequences (DNA from healthy cells) are also amplified. It was surprisingly found that, in the method according to the invention, the number of false positives is significantly reduced as a result compared with a (conventional) methyl-specific PCR (MS-PCR) using methyl-specific primers (MSP). To this end, for each primer pair the bias is first determined according to the MgCl2 concentration, annealing temperature and number of cycles. In this way, considerably higher diagnostic specificities are achieved for distinguishing between healthy patients and those with tumours, without reducing the analytical sensitivity. A high diagnostic specificity, i.e. a low rate of false positives, is critical in deciding whether a test procedure can be used for screening for tumour diseases or precautionary examinations. In the method according to the invention, partially methylated DNA (known as heterogeneously methylated epialleles) are also advantageously amplified. In step b) (pre-amplification), the reaction conditions, such as MgCl2 concentration and annealing temperature, are preferably selected such that the majority of the methylated sequences are copied regardless of how many unmethylated sequences there are in the sample being tested, and only unmethylated sequences are copied when there are no methylated DNA sequences present.
The term “target of interest” or target sequence also includes regulatory sequences outside of an open reading frame (ORF)
Advantageously, it has been found that, in the method according to the invention, the preferred pre-amplification of methylated sequences is also possible in samples in which there are high levels of background DNA (unmethylated DNA). In the method according to the invention, this DNA does not pose a problem. As a result, the method according to the invention is also suitable for analysing tumour DNA in bodily fluids, smears or tissue samples (“circulating free tumour DNA”, cf-tumour DNA).
Generally, methylated DNA sequences are tumour-specific and are thus amplified to a greater extent in step b) than the unmethylated DNA sequences. This is the case, for example, in the targets of interest PLA2R1, RASSF1A, GSTP1, AOX1, SERPINE-1 and thrombomodulin preferably studied in the following. However, there are also cases, e.g. the MGMT gene in glioblastoma [15], in which the unmethylated DNA sequences are tumour-specific and the methylated DNA sequences are found in the healthy tissue. In these cases, the unmethylated DNA sequences are preferably amplified to a greater extent in step b) than the methylated DNA sequences.
In step c), quantification is carried out by means of digital PCR (also referred to as dPCR below). In the known digital PCR methods, the DNA used undergoes limiting dilution in such a way that no DNA molecules or precisely one DNA molecule is present in as many compartments as possible (Poisson distribution). The inventors have now surprisingly discovered that increasing the amount of DNA used in the dPCR beyond the Poisson distribution (e.g. with 10,000 compartments, more than 80,000 DNA copies are analysed in the dPCR, i.e. at a copy per compartment (CPC) rate of >8) significantly improves the specificity and the distinction between healthy samples and malignant tumour diseases. According to the prior art, a Poisson distribution is present for the dPCR when the CPC is <8 since otherwise there are no compartments without DNA copies available to form the basis for the calculations [9].
The combination of steps b) and c) (also referred to as BBPA-dPCR in the following) advantageously achieves considerably higher analytical and diagnostic sensitivities, and the method according to the invention also surprisingly makes it possible to draw much more reliable conclusions as to whether or not there is a malignant tumour. As a result, the method is suitable for early detection screening (precautionary examinations). A further advantage of the method according to the invention is that it makes it possible to distinguish between benign and malignant tumours and to detect a minimal residual disease (MRD), thereby allowing the treatment and disease progression to be monitored. The principle of the method according to the invention is summarised in FIG. 1 on the basis of embodiments.
The inventors' preliminary studies surprisingly showed that, when an optimum annealing temperature was selected in the BBPA, the methylation level in the targets of interest studied in samples from healthy subjects (as determined in the subsequent dPCR) approached 0% as the number of PCR BBPA cycles increased, and that, at an appropriate annealing temperature, the methylation level in samples from female breast cancer patients and prostate cancer patients approached 100% when a suitable MgCl2 concentration and number of cycles were selected in the BBPA (FIGS. 2-4, 7 and 8, Tables 2-5). This unexpected phenomenon is not yet fully understood, but it can be used to significantly improve the distinction between healthy samples, i.e. with no fc-tumour DNA, and diseased samples, i.e. with fcT-DNA detected accordingly. In principle, such a clear-cut distinction makes it possible to give a yes/no answer to the question of whether or not there is tumour-specific DNA and therefore a tumour disease present. In principle, this method makes it possible to specifically detect just one single tumour DNA molecule against a large background of normal DNA.
The greater the proportion of methylated sequences, the greater the likelihood of there being a malignant tumour disease, in particular in the advanced stage.
According to the prior art, digital PCR alone and the previously known PCR-based techniques alone (MS-qPCR or PCR followed by melting curve analysis (MS-HRM)) are not able to reliably distinguish between patients with tumours and healthy patients (with no tumours) using bodily fluid samples (liquid biopsy tests). For example, in the event of results that are very close to one another, i.e. in terms of the proportion of fcT-DNA compared with the proportion of normal wild type DNA, and no reliable distinction can be drawn between healthy patients and those with tumours, no further distinctions can be made by means of MS-HRM or dPCR alone unless additional DNA is added to the tests. However, this is often not possible when liquid biopsy materials, e.g. serum, plasma, urine, cerebrospinal fluid or smears, are used. By contrast, increasing the number of BBPA cycles in the BBPA-dPCR technique according to the invention makes it easier to clearly distinguish between healthy and diseased samples. This is particularly significant when distinguishing between benign hyperplasia, e.g. benign prostatic hyperplasia (BPH), and malignant diseases such as prostate cancer, as demonstrated in the embodiments on the basis of cell cultures and serum samples from prostate cancer patients.
In step b), the methylated and unmethylated DNA sequences of the targets of interest are preferably copied using a correspondingly high number of PCR cycles (preferably from 10 to 50 cycles), and are then quantified in the dPCR (step c)) either directly or after the amplified material has been slightly pre-diluted if the number of BBPA cycles is high.
Next, the individual steps of the method and preferred embodiments will be explained in more detail:
Prior to step a), DNA is preferably isolated (step a′)) using known methods. A person skilled in the art also knows to carry out bisulphite conversion. The person skilled in the art can use commercially available kits for both steps.
In principle, the isolated sample can be a tissue sample. Advantageously, however, the method according to the invention is also suitable for detecting methylated tumour DNA in bodily fluids (liquid biopsy), e.g. full blood, serum, plasma, urine, cerebrospinal fluid, sputum, bronchial washing, semen, nipple discharge, vaginal secretion (smear) or lymph, particularly preferably serum, plasma or urine.
In step b) (BBPA), methylated and corresponding unmethylated DNA sequences of the same gene portion are copied by means of PCR such that the methylated DNA sequences (when specific for tumours) or unmethylated DNA sequences (when specific for tumours) are amplified to a greater extent.
Unlike the prior art, in which it is recommended to use primer pairs having at most two CpG sites, in some cases three CpG sites, that are all as close as possible to the 5′ end but preferably not located at the 3′ end of the primer sequences [4, 16-19], primers that together contain up to seven CpG sites in their sequences can be used according to the invention, in particular in combination with suitable MgCl2 concentrations and annealing temperatures. In the process, the CpG sites can be distributed over the entire primer sequence, preferably so close to the 3′ end that a cytosine of a CpG dinucleotide sequence is located directly at the 3′ end. In this way, tumour DNA can be detected in a significantly more sensitive and at the same time more specific manner, i.e. without an increase in false-positive signals, even when there is a high proportion of normal DNA (wild-type DNA) in the sample being tested, which is often the case in human bodily fluids, smears or tissue samples. In addition, primers can also be designed for testing CpG-rich gene sequences that would otherwise not have been able to be tested.
Since the primers and in particular also the reaction conditions (annealing temperature always at MgCl2 concentrations optimised accordingly in the reaction buffer) are selected such that they amplify both methylated and unmethylated DNA sequences, i.e. they are methylation-independent primers (MIP), when optimised reaction conditions are selected, the primers enter into competition reactions for methylated and unmethylated DNA molecules, thereby preventing false-positive signals, unlike when methylation-specific primers (MSP) are used. As well as higher specificity, methylated DNA copies can thus be identified in a considerably more sensitive and specific manner against a large background of unmethylated DNA copies. In addition, heterogeneously methylated DNA fragments (epialleles) are also quantified as well as homogeneously methylated fragments, thereby allowing for a more clear-cut differentiation between tumour diseases and healthy samples.
Preferably, the PCR conditions (in particular primer sequences, magnesium concentration, annealing temperature and in particular also the number of cycles) are set such that the bias is optimised in favour of the unmethylated DNA sequences, while unmethylated DNA sequences are also amplified at the same time.
In step b), magnesium concentrations (final concentrations) of from 0.5 to 15 mmol/l, preferably from 1 to 10 mmol/l, particularly preferably from 2 to 5 mmol/l, in particular from 2 to 4 mmol/l, particularly preferably from 2.5 to 3.5 mmol/l are preferably selected for the PCR.
The inventors have discovered that the bias can be shifted in favour of the methylated DNA sequences by a high annealing temperature and high 5′-CG-3′ content. When the annealing temperature, MgCl2 concentration, number of cycles and primer design are adapted to one another appropriately, the specificity and sensitivity of the method can advantageously be increased such that a liquid biopsy can be used for early tumour screening (precautionary examinations) and to detect a minimal residual disease (MRD). If methylated sequences are to be preferably pre-amplified over unmethylated sequences, high annealing temperatures and lower MgCl2 concentrations (though only so low as to ensure unmethylated DNA sequences are still pre-amplified to a minor extent, thus preventing false-positive signals) are preferably selected in addition to the 5′-CG-3′-containing primer sequences. If unmethylated sequences are to be preferably pre-amplified over methylated sequences, in addition to the primer design (no 5′-CG-3′-containing sequence as far as possible, or at most two such sequences) the annealing temperature and MgCl2 concentration can be adjusted accordingly in line with preliminary tests described below.
The primers are preferably selected in step b) such that they contain from zero to seven 5′-CG-3′ dinucleotide sequences per primer pair, preferably from two to six, preferably from three to five, particularly preferably from one to at most three, more preferably either one or two 5′-CG-3′ dinucleotide sequences per primer pair.
It has surprisingly also been found that the diagnostic sensitivity of the method can be increased further, i.e. a greater number of pathological results where there is actually a tumour disease, when, per target of interest, two or more (different) primer pairs that preferably all include the sequences detected by the probes in dPCR are used separately or simultaneously in the pre-amplification.
The annealing temperatures are preferably above 40° C., in particular above 45° C., and are preferably between 50 and 72° C., preferably between 53 and 70° C., particularly preferably up to 63° C. Like the MgCl2 concentrations and the optimum number of cycles, the optimum annealing temperatures are preferably determined empirically for each primer pair.
The bias is optimised in favour of the methylated DNA sequences preferably by empirically adjusting the primer sequences and annealing temperatures, preferably together with the MgCl2 concentrations, following the aforementioned selection rules. For each target of interest or each gene sequence, the PCR conditions (in particular primer selection and annealing temperature, together with the MgCl2 concentration and number of cycles) are preferably determined using a sample having a known ratio of methylated DNA to unmethylated DNA (FIGS. 34-37 and Tables 21 and 22). If no bias is achieved using primers without 5′-CG-3′ dinucleotide sequences, or the bias is only achieved at high annealing temperatures (e.g. above 70° C.), one 5′-CG-3′ dinucleotide sequence is added; if this is insufficient, at most two, three or four 5′-CG-3′ dinucleotide sequences are added (i.e. either one 5′-CG-3′ dinucleotide sequence added to the forward and reverse primer sequence or from two to four 5′-CG-3′ dinucleotide sequences added to the forward or reverse primer sequence). At low MgCl2 concentrations (e.g. below 1.0 mmol/l), three 5′-CG-3′ dinucleotide sequences are preferably added; if this is insufficient, at most eight, preferably at most seven 5′-CG-3′ dinucleotide sequences are added per primer pair. In this case, it is irrelevant whether the 5′-CG-3′ dinucleotide sequences are found at the 5′ end, as is recommended in previous literature [4, 16-19]. On the contrary, the sensitivity of tests on samples containing a high background of normal wild-type DNA can be increased using primers in which the 5′-CG-3′ dinucleotide sequences are located particularly close or directly at the 3′ end of the primers [FIGS. 38-45 and Tables 27-30]. In addition to higher analytical sensitivity, it is thus also possible to advantageously construct primers for gene portions that are characterised by a high density of 5′-CG-3′ dinucleotide sequences and have previously not been used for testing in accordance with the current literature recommendations [4, 16-19].
In particular, the number of PCR cycles in step b) is dependent on the starting concentration of the DNA in the sample being tested. Depending on the amount of DNA available, cycle numbers of from 5 to 50, preferably from 10 to 50, particularly preferably at least 15 and/or up to 40 are selected.
In principle, the method according to the invention can be used for any target of interest being tested. Preferably, the methylation level in DNA sequences of from three to five, preferably of up to six targets of interest are analysed in the method according to the invention. For this purpose, step b) is preferably carried out as multiplex PCR, i.e. the primer pairs for pre-amplifying the target sequences are adapted to one another such that they have approximately the same annealing temperature and do not hybridise with one another. Preferably, the primer pairs for pre-amplifying the target sequences in step b) are selected such as to produce the highest analytical and diagnostic sensitivities and specificities at the same annealing temperature, MgCl2 concentration and number of cycles.
The two-stage method (BBPA-dPCR) is advantageous in that the method has a significantly higher diagnostic sensitivity and in particular higher specificity too compared with previous methods such as dPCR, MSP or MS-HRM, meaning that in principle a single tumour molecule can be detected against a large background of normal DNA (wild-type DNA). Therefore, serum samples can also be tested in addition to plasma, without having to carry out any special pre-analysis. The analytical and diagnostic sensitivity of the novel method is ultimately limited only by the sample being tested actually containing a single tumour DNA molecule that has the target gene sequence and can be transferred to the pre-amplification.
False-positive signals are prevented by simultaneously pre-amplifying unmethylated DNA fragments, even if this is a result of significantly lower efficiency towards methylated DNA sequences or, as surprisingly found, with higher efficiency towards methylated DNA sequences in samples from subjects not having tumour diseases. This is presumably due to the primers entering into competition reactions for the target sequences. In addition, the determined relative methylation levels in the targets of interest can be compared between individual patient samples despite the PCR bias since the level of the bias is the same for each sample tested at constant PCR conditions (constant annealing temperature, MgCl2 concentration and number of cycles). Owing to the aforementioned advantages and the high diagnostic specificity achieved in the novel method, it is also possible to distinguish between benign hyperplasia and malignant diseases. Preferably, this is beneficial for the differential diagnosis of benign prostatic hyperplasia (BPH), prostatitis and prostate cancer (PCa) diseases since a prostate tissue biopsy is indicated when PSA values are elevated (the critical range is between 2.0 and 15.0 mg/ml; the reference range is from <2.5-4.0 mg/ml).
The higher the proportion of methylated sequences (in particular when specific to tumour DNA), the greater the likelihood of there being an advanced malignant tumour disease. If unmethylated sequences are tumour-specific, the opposite applies.
Depending on the selected primers and PCR conditions (annealing temperature, magnesium chloride concentration and number of cycles) in the pre-amplification, the percentages (fractional abundance) of methylated sequence copies pointing to the absence of a malignant disease in the form of a reference range (healthy normal range) are determined. In the embodiments, for example, a malignant tumour disease is preferably indicated by a proportion of methylated sequences of >2% in PLA2R1 (preferably 168 bp fragment), >0.1% in RASSF1A (preferably 117 bp fragment), >2% in GSTP1 (preferably 120 bp fragment) and >0.05% in GSTP1 (preferably 116 fragment). For each target sequence (and specific PCR conditions), these cut-off values between healthy and diseased samples are determined by also carrying out controls for healthy samples (e.g. DNA isolated from healthy epithelial cells from the prostate (PrEC), the breast (HMEC, MCF10A cell line) or other tissues to be tested) and diseased samples (e.g. DNA isolated from malignant LNCAP, PC-3 and DU145 prostate cells, malignant MCF-7, Cal-51, UACC-812, BT-474, MDA-MB-453 and MDA-MB231 breast tissue cells).
Patients are preferably classified as tumour patients when they present elevated values for homogeneously or heterogeneously methylated epialleles for at least one, preferably two, or even more target gene sequences.
In step c), quantification is carried out by means of digital PCR, although, by contrast to the conventional dPCR, the number of pre-amplified DNA copies used is outside the normal Poisson distribution.
Surprisingly, “overloading” the digital PCR in this way with quantities of DNA copies above a CPC value of 8 leads to increased specificity and thus considerably better distinctions between healthy samples (with no tumour disease) and malignant tumour diseases (see Table 6 in conjunction with Tables 4 and 5). For example, the quantity of samples from healthy subjects used in the dPCR produced a CPC value of 560, and the CPC value for prostate cancer patients was 1938—the values at which the best distinction (i.e. as low a value as possible for healthy samples and as high a value as possible for diseased samples) between healthy and diseased samples was achieved (Table 5).
For this purpose, the DNA pre-amplified in step b) is preferably distributed into the compartments for the digital PCR in non-diluted or slightly diluted form (e.g. up to 1:1000 after 50 cycles). Preferably, such a quantity of DNA is used in the digital PCR for there to be at least one DNA molecule in each compartment, preferably at least five molecules. Preferably, the quantity of DNA used is double that permitted by the Poisson distribution and statistics. Preferably, there are an average of at least 10, preferably at least 20, more preferably at least 50, particularly preferably at least 100 DNA molecules per compartment. In this case, this copies per compartment number (CPC) denotes the average number (arithmetic mean) of dsDNA molecules per compartment.
The number of copies of pre-amplified DNA to be used in the dPCR can be calculated as follows:Number of copies=CPC*number of compartments
Therefore, for a preferred CPC of 8 and 5,000 compartments for example, 40,000 DNA copies are required; if there are 100,000 compartments, the number of copies required is accordingly 800,000. For a particularly preferred CPC of 50 and if there are 100,000 compartments, 5×106 copies must be input into the dPCR. When using the RainDrop (RainDance Technologies) digital system, for example, which has up to 1 million compartments (droplets), a preferred CPC of 10 means 1×107 DNA copies must be used in the dPCR; a particularly preferred CPC of 50 means 5×107 DNA copies must be used.
The methylated and unmethylated DNA sequences are quantified in step c) using probes that specifically hybridise with methylated regions of the pre-amplified target sequences or with the corresponding unmethylated regions.
For each target of interest, the probes for the methylated DNA sequences preferably comprise a total of at least three, preferably at least four, and up to eight, preferably up to seven 5′-CG-3′ dinucleotides. For each gene, the probes for the unmethylated DNA sequences preferably comprise a total of at least three, preferably four, and up to eight, preferably up to seven 5′-CA-3′ dinucleotides. Alternatively, the probes can be used to detect the complementary strand in the amplified double-strand DNA molecule. This is done either separately or together with the probe for the coding strand. To detect the complementary strand, the probes for the unmethylated DNA sequences preferably comprise three, preferably four, and up to seven 5′-TG-3′ dinucleotides (instead of the 5′-CA-3′ dinucleotides). The number of 5′-CG-3′ dinucleotides in the probes for the methylated DNA sequences preferably corresponds to the number of 5′-CA-3′ dinucleotides (or 5′-TG-3′ dinucleotides) in the probes for the unmethylated DNA sequences of the same gene. Per target of interest, the aforementioned number of 5′-CG-3′ or 5′-CA-3′ (or 5′-TG-3′) dinucleotides is either contained in one probe or distributed over a plurality of probes (in particular when it is not possible to design a probe comprising all the methylation sites due to the sequence in the target of interest). Where there are two probes for one target of interest, both probes for the methylated sequences preferably each contain three or four 5′-CG-3′ dinucleotides and both probes for the unmethylated sequences preferably comprise three or four 5′-CA-3′ (or 5′-TG-3′) dinucleotides.
Otherwise, the digital PCR is carried out and quantified according to the usual methods. The compartments are preferably oil droplets (droplet digital PCR, ddPCR). Alternatively, the compartments are preferably arranged on a chip. However, more than one amplification cycle is preferably carried out in digital PCR, preferably at least 5, preferably at least 15, more preferably from 20 to 50, particularly preferably from 30 to 45, more preferably up to 40 cycles. For the quantification, the compartments in which amplification has taken place and with which the probes for the methylated and unmethylated DNA sequences hybridise are counted. The same primers used for the BBPA in step b) can also be used as primers for the dPCR. Alternatively and preferably, nested primers that bond to other sites on the pre-amplified sequence are used. A nested PCR can further increase the specificity. In this case, the primers can be selected according to the standard rules and the selection rules mentioned in step b) do not necessarily have to be followed. Preferably, however, the primers are selected according to the same rules as in step b).
Within the meaning of the invention, therefore, digital PCR (dPCR) is understood to be a PCR in a large number of separate compartments, preferably using a volume in the picolitre or nanolitre range. dPCR is distinguished by the compartments being quantified according to a digital result (amplification: yes or no). By counting a large number of reaction compartments (high-throughput screening preferably using from 10,000 to 100,000 compartments per PCR), statistical significance is obtained. The proportion of reaction chambers in which amplification was successful is proportional to the quantity of DNA used for the amplified DNA sequence; this is used to determine the quantity.
The probes are preferably fluorescently tagged, the probes for methylated and unmethylated sequences preferably being provided with different fluorescent markers. The fluorescent label is preferably attached to one end of the probe (preferably the 5′ end). At the other end, there is preferably a quencher that is adapted to the fluorescent label and suppresses the fluorescent signal. As a result of the hybridisation with the amplified DNA, or following hybridisation and subsequent polymerase action, the quench effect is lifted and the fluorescent signal can be detected. Fluorescent labels and quenchers of this kind are well known to a person skilled in the art and are commercially available for any probe sequences.
If suitable multicolour fluorescence detection systems are available, the digital CPR is preferably also carried out as multiplex PCR. For the quantification, different colour probes are preferably used for each amplified target of interest. In this case, the probes are preferably designed such that they have a comparable hybridisation temperature.
Alternatively, the probes are used and assessed in separate batches in the dPCR. This is also applicable when the probes have different hybridisation temperatures.
If a plurality of probes are used per target of interest, the probes can have the same fluorescent markers and the signal can be integrated. Alternatively, the two aforementioned alternatives for multiplex PCR are used.
For the pre-amplification in step b), primers that amplify methylated and unmethylated DNA sequences in the genes PLA2R1, RASSF1A and GSTP1 are preferably used (methylated DNA being amplified to a greater extent than unmethylated DNA). In step c), the methylation of these targets of interest is quantified.
As shown in the embodiments (see in particular Tables 7-11), selecting these three targets of interest advantageously makes it possible to distinguish between healthy and diseased samples for all tumours tested (prostate cancer, breast cancer, ovarian cancer and renal cell cancer).
To pre-amplify methylated and unmethylated DNA sequences for the genes PLA2R1 (phospholipase A2 receptor 1, HGNC Ace. HGNC 9042, Ensembl: ENSG00000153246), RASSF1A (Rass association (RalGDS/AF-6) domain family member 1, HGNC Ace. HGNC 9882, Ensembl: ENSG00000068028) and GSTP1 (glutathione S-transferase pi 1, HGNC Acc HGNC 4638, Ensembl: ENSG00000084207), the following primer pairs are preferably used in step b):
SEQSEQTargetForward (5′->3′)IDReverse (5′->3′)IDPLA2R1GGGGTAAGGAAGGTGGAGAT1ACAAACCACCTAAATTCTAATAAACAC2(168 bp) RASSF1AGTTTGTTAGCGTTTAAAGTTAG3AATACGACCCTTCCCAAC4(117 bp) GSTP1GTGAAGCGGGTGTGTAAGTTT5TAAACAAACAACAAAAAAAAAAC6(120 bp)
Optionally, instead of the PLA2R1 (168 bp) forward primer (SEQ ID No 1), the forward primer according to SEQ ID No 7 and/or 53 is used, and/or instead of the reverse primer (SEQ ID No 2), one of the following reverse primers (SEQ ID No 8, 9, 10 or 11) is used:
TargetForward (5′->3′)SEQ IDReverse (5′->3′)SEQ IDPLA2R1GGAAGGTGGAGATTACGG7GCGAATTTACAACGAACAAC8(133 bp) PLA2R1GGGGTAAGGAAGGTGGAGAT53AATAAACACCGCGAATTTACAAC9(150 bp) PLA2R1ACCTAAATTCTAATAAACACCGC10(161 bp) PLA2R1CCTAAATTCTAATAAACACCGC11(160 bp)
Optionally, instead of the GSTP1 (120 bp) forward primer (SEQ ID No 5), the forward primers according to SEQ ID No 91, 93, 95 or 97 are used, and/or instead of the reverse primer (SEQ ID No 6), the reverse primers according to SEQ ID No 92, 94, 96 or 98) are used:
TargetForward (5′->3′)SEQ IDReverse (5′->3′)SEQ IDGSTP1CGTAGCGGTTTTAGGGAATTT91TCCCCAACGAAACCTAAAAA92(114 bp) GSTP1ATCGTAGCGGTTTTAGGGAA93TCCCCAACGAAACCTAAAAA94(116 bp) GSTP1TGTAAGTTTCGGGATCGTAGC95TCCCCAACGAAACCTAAAAA96(129 bp) GSTP1GTGTGTAAGTTTCGGGATCG97TCCCCAACGAAACCTAAAAA98(132 bp)
Furthermore, instead of the GSTP1 (120 bp) forward primer (SEQ ID No 5), the forward primer according to SEQ ID No 12 is optionally used, and/or instead of the reverse primer (SEQ ID No 6), the reverse primer according to SEQ ID No 13 is used:
SEQSEQTargetForward (5′->3′)IDReverse (5′->3′)IDGSTP1GTTCGGTTAATATGGT12ACCCAAACTAAAATACAAT13(171 bp)GAAAAC
This primer pair is particularly preferable when the GSTP1 probes having 4 CpG sites (preferably SEQ ID No 45 and 46 or complementary sequences) are used subsequently in the dPCR, and/or when the GSTP1 probes having 5 CpG sites (preferably SEQ ID No 47 and 48 or complementary sequences) are used.
To further increase the significance of the method according to the invention or its scope of application (other tumours), in a preferred embodiment methylated and unmethylated sequences in the gene(s) AOX-1 (aldehyde oxidase 1, HGNC Acc HGNC 553, Ensembl: ENSG00000138356) and/or SERPINE-1 (serpin peptidase inhibitor, HGNC Ace HGNC 8583, Ensembl: ENSG00000106366) and/or thrombomodulin (HGNC THBD, HGNC 11784, Ensembl: ENSG00000178726) and/or septin-9 (SEPT9, HGNC 7323 Ensembl: ENSG00000184640) are additionally pre-amplified and the methylation of these genes is quantified in step c).
Preferably, the primers for pre-amplifying these targets of interest are selected as follows:
SEQSEQTargetForward (5′->3′)IDReverse (5′->3′)IDAOX1TGGGTTGGATTTTAGGTTTTAG14CTCACCTTACGACCGTTC15(180 bp) SERPINE1AGAGCGTTGTTAAGAAGA16CTCCTACCTAAAATTCTCAAAA17(123 bp)
Optionally, instead of the AOX1 (180 bp) forward primer (SEQ ID No 14), the forward primer according to SEQ ID No 18 is optionally used, and/or instead of the reverse primer (SEQ ID No 15), the reverse primer according to SEQ ID No 19 is used:
SEQSEQTargetForward (5′->3′)IDReverse (5′->3′)IDAOX1GTTGGATTTTAGGTTTT18GCCCGATCCATTATAAT19(138 bp)AGTAAGATC
This primer pair is particularly preferable when the AOX1 probes having 4 CpG sites (preferably SEQ ID No 39 and 40 or complementary sequences) are used subsequently in the dPCR, and/or when the AOX1 probes having 5 CpG sites (preferably SEQ ID No 41 and 42 or complementary sequences) are used.
For the quantification per dPCR, probes selected from the following sequences are preferably used in step c):
SEQSEQTargetmethylated (5′->3′)IDunmethylated (5′->3′)IDPLA2R1CCCAACTACTCCGCGACGCAA20AACCCAACTACTCCACAACACAAA21(3 CpG) PLA2R1CAACTACTCCGCGACGCAAACG22AACCCAACTACTCCACAACACAAACA23(4 CpG) RASSF1ACGCCCAACGAATACCAACTCCCG24CACCCAACAAATACCAACTCCCACAA25(3 CpG) RASSF1ACGCCCAACGAATACCAACTCCCGCG54CACCCAACAAATACCAACTCCCACAACTC55(4 CpG) GSTP1CGCAACGAAATATACGCAAC56CACAACAAAATATACACAAC57(3 CpG) GSTP1ACGAACTAACGCGCCGAAAC58ACAAACTAACACACCAAAAC59(4 CpG) GSTP1CGATCTCGACGACTCACTACAACC45CAATCTCAACAACTCACTACAACCTC46(3 CpG) GSTP1CGCGATCTCGACGACTCACTACAA47CACAATCTCAACAACTCACTACAACCT48(4 CpG)
These are complementary to the coding strand.
Alternatively, each (complementary) template strand can also be quantified either separately or together with the coding strand in one batch. The following (complementary) probes are suitable and preferable for this purpose:
SEQSEQTargetmethylated (5′->3′)IDunmethylated (5′->3′)IDPLA2R1TTGCGTCGCGGAGTAGTTGGG60TTTGTGTTGTGGAGTAGTTGGGTT26(3 CpG) PLA2R1CGTTTGCGTCGCGGAGTAGTTG27TGTTTGTGTTGTGGAGTAGTTGGGTT28(4 CpG) RASSF1ACGGGAGTTGGTATTCGTTGGGCG29TTGTGGGAGTTGGTATTTGTTGGGTG30(3 CpG) RASSF1ACGCGGGAGTTGGTATTCGTTGGGCG31GAGTTGTGGGAGTTGGTATTTGTTGGGTG32(4 CpG) GSTP1GTTGCGTATATTTCGTTGCG33GTTGTGTATATTTTGTTGTG34(3 CpG) GSTP1GTTTCGGCGCGTTAGTTCGT35GTTTTGGTGTGTTAGTTTGT36(4 CpG) GSTP1GGTTGTAGTGAGTCGTCGAGATCG49GAGGTTGTAGTGAGTCGTCGAGATCG50(3 CpG) GSTP1TTGTAGTGAGTCGTCGAGATCGCG51AGGTTGTAGTGAGTCGTCGAGATCGCG52(4 CpG)
If methylated and unmethylated sequences of the gene(s) AOX-1 and/or SERPINE-1 are pre-amplified, probes selected from the following sequences and complementary sequences are particularly preferably used in step c) to quantify the methylation of these genes:
SEQSEQTargetmethylated (5′->3′)IDunmethylated (5′->3′)IDAOX1ACTCGAACGCCCGATCCATTATAA37ACAACTCAAACACCCAATCCATTATAA38(3 CpG) AOX1CGCTAATTCGAAAACCCGAAACGA39CACTAATTCAAAAACCCAAAACAA40(4 CpG) AOX1CGCGCTAATTCGAAAACCCGAAACGA41CACACTAATTCAAAAACCCAAAACAA42(5 CpG) SERPINE1CGATTAACGATTCGTCCTACTCTAACG43CAATTAACAATTCATCCTACTCTAACA44(4 CpG)
Alternatively or additionally, the following additional primer pairs can be used in step c):
SEQSEQTargetForward (5′->3′)IDReverse (5′->3′)IDRASSF1AGCGTTTGTTAGCGTTTAAAG61AACCGAATACGACCCTTC62(124 bp) AOX1GTTGGATTTTAGGTTTTAGTAAG63GCCCGATCCATTATAATATC64(138 bp) AOX1GGATTTTAGGTTTTAGTAAGTTTC65GCCCGATCCATTATAATATCCG66(135 bp) AOX1GATTTTAGGTTTTAGTAAGTTTCG67(134 p) AOX1TTTTAATTAAGGTTTTTTTCGTCG99CCCGATCCATTATAATATCCG100(171 p) SERPINE1CGTTGTTAAGAAGATTTATAC68TAAACCCGAAATAAAAAATTAAA69(119 bp) TMGGTCGATTCGTATGTTAGA70AACCGTACCGAAACAAAA71(125 bp) TMGTTTGGGTTGGGACGGATA72AAAAACCAAAACCCCAAACA73(144 bp) TMGTTTGGGGTTTTGGTTTTTG74GCAATCCGTCGCAAATCTAA75(166 bp) TMCAATCCGTCGCAAATCTAAC76(165 bp) TMTTTGTGTTTTTTTGTTTCGGTAC101CACCCGACTACGACTCTACG102(160 bp) TMACCCGACTACGACTCTACGA103(159 bp) RASSF1ATTTAGTTTGGATTTTGGGGGA104CTAACTTTAAACGCTAACAAA105(86 bp) RASSF1AGTTTGGATTTTGGGGGAGC106ACTTTAAACGCTAACAAACG107(82 bp) RASSF1ATTTGGATTTTGGGGGAGCG108CGCTAACTTTAAACGCTAAC109(82 bp) RASSF1ATTTAGTTTGGATTTTGGGGGAG110CGCTAACTTTAAACGCTAACAAA111(86 bp) TMGGTCGATTCGTATGTTAGA126AACCGTACCGAAACAAAA127(125 bp) TMTAGCGGTAAGAAGTGTTTG128(83 bp) TMTACGGTTTTGTCGTAGTG129CCCAAACATATTACCCAAAC130(70 bp) TMGGAGAGGTTGTCGTTATC131CCCAAACATATTACCCAAAC132(113 bp) TMACCCCAAACATATTACCC133(115 bp) TMGTCGAGTACGATTGTTTC134ACGCACTATCATTAAATAACC135(99 bp) TMCGGTGGTTGTCGATGTTA136CCGCAACCGAATAACAAC137(97 bp) TMTTGCGGGGTTATTTAATG138CAACCGAATAACAACTACA139(125 bp) Septin-9GCGATTCGTTGTTTATTAG140ATCCGAAATAATCCCATC141(72 bp) Septin-9CGGTTAGTTTTGTATTGTAG142(169 bp) Septin-9CGGGGTTGTTTTGTTTAAG143CCAACACCGACAATCAAA144(94 bp)
The AOX1 primer pairs (SEQ ID No 63 and 64 or 65 and 66 or 65 and 67 or 99 and 100) are particularly preferable when the AOX1 probes having 4 CpG sites (preferably SEQ ID No 77 and 78 or 79 or complementary sequences) are used subsequently in the dPCR, or when the AOX1 probes having 5 CpG sites (preferably SEQ ID No 112 and 113 or 112 and 114 or complementary sequences) are used.
The thrombomodulin (TM) primer pairs (SEQ ID No 70 and 71 or 72 and 73 or 101 and 102 or 101 and 103) are particularly preferable when the TM probes having 3 CpG sites (preferably SEQ ID No 80 and 81 or complementary sequences) are used subsequently in the dPCR, and the TM primer pairs (SEQ ID No 74 and 75 or 74 and 76) are particularly preferable when the TM probes having 4 CpG sites (preferably SEQ ID No 82 and 83 or complementary sequences) are used.
The thrombomodulin (TM) primer pairs (SEQ ID No 126 and 127 or 127 and 128) are particularly preferable when the TM probes having 3 CpG sites (preferably SEQ ID No 145 and 146 or complementary sequences) are used subsequently in the dPCR, and the TM primer pairs (SEQ ID No 129 and 130 or 131 and 132) are particularly preferable when the TM probes having 5 CpG sites (preferably SEQ ID No 147 and 148 or complementary sequences) are used. The thrombomodulin (TM) primer pairs (SEQ ID No 131 and 133) are particularly preferable when the TM probes having 5 CpG sites (preferably SEQ ID No 149 and 150 or complementary sequences) are used subsequently in the dPCR, the TM primer pairs (SEQ ID No 134 and 135) are particularly preferable when the TM probes having 3 CpG sites (preferably SEQ ID No 151 and 152 or complementary sequences) are used, the TM primer pairs (SEQ ID No 136 and 137) are particularly preferable when the TM probes having 5 CpG sites (preferably SEQ ID No 153 and 154 or complementary sequences) are used, and the TM primer pairs (SEQ ID No 138 and 139) are particularly preferable when the TM probes having 4 CpG sites (preferably SEQ ID No 155 and 156 or 157 and 158 or complementary sequences) are used.
The septin-9 primer pairs (SEQ ID No 140 and 141) are particularly preferable when the TM probes having 4 CpG sites (preferably SEQ ID No 159 and 160 or complementary sequences) are used subsequently in the dPCR, the septin-9 primer pairs (SEQ ID No 141 and 142) are particularly preferable when the TM probes having 4 CpG sites (preferably SEQ ID No 161 and 162 or complementary sequences) are used, and the septin-9 primer pairs (SEQ ID No 143 and 144) are particularly preferable when the TM probes having 2 CpG sites (preferably SEQ ID No 163 and 164 or complementary sequences) are used.
The RASSF1A primer pairs (SEQ ID No 104 and 105 or 106 and 107 or 108 and 109 or 110 and 111) are particularly preferable when the RASSF1A probes having 4 CpG sites (preferably SEQ ID No 115 and 116 or 115 and 117 or 115 and 116 or complementary sequences) are used subsequently in the dPCR.
For the quantification per dPCR, probes selected from the following sequences are preferably used in step c):
SEQSEQTargetmethylated (5′->3′)IDunmethylated (5′->3′)IDAOX1CGCTAATTCGAAAACCCGAAACGA77CACTAATTCAAAAACCCAAAACAA78(4 CpG) AOX1CACTAATTCAAAAACCCAAAACAAAAA79(4 CpG) AOX1CGCGCTAATTCGAAAACCCGAAACGA112CACACTAATTCAAAAACCCAAAACAA113(5 CpG) AOX1CACACTAATTCAAAAACCCAAAACAAAAA114(5 CpG) RASSF1ACGCGAACCGAACGAAACCAC115CACAAACCAAACAAAACCAC116(4 CpG) RASSF1ACACAAACCAAACAAAACCACAAA117(4 CpG) RASSF1AAAACACAAACCAAACAAAACCACAAA118(4 CpG) TMACGCCGATAACGACAACCTCT80AAAAAGCAGATAAAGACAACCTCT81(3 CpG) TMCCGACTACGACTCTACGAATACGAA82CAGACTAAGACTCTAAGAATAAGAAAAAC83(4 CpG) TMACGCCGATAACGACAACCTCT145AAGCAGATAAAGACAACCTCT146(3 CpG) TMCGCCGCGTACAAACGCCGAA147AGCAGAGTACAAAAGCAGAA148(5 CpG) TMAACGCGCCGCGTACAAACGC149AAAGAGCAGAGTACAAAAGC150(5 CpG) TMCGCAATCCGTCGCAAATCTAACT151AGCAATCAGTAGCAAATCTAACT152(3 CpG) TMAACGCCGACGACCAACGCCG153AAAGCAGAAGACCAAAGCAG154(5 CpG) TMAAAACGCCGACGACCAACGC155AAAAAGCAGAAGACCAAAGC156(4 CpG) TMAAAACGCCGACGACCAACGCC157AAAAAGCAGAAGACCAAAGCC158(4 CpG) Septin-9CGTTAACCGCGAAATCCGACATAAT159AGTTAACAGAGAAATCAGACATAAT160(4 CpG) Septin-9CGTTAACCGCGAAATCCGACATAATAA161AGTTAACAGAGAAATCAGACATAATAA162(4 CpG) Septin-9AAACGCACGCACTCACAAACT163AAAAGCAAGCACTCACAAACT164(2 CpG)
These are complementary to the coding strand.
Alternatively, each (complementary) template strand can also be quantified either separately or together with the coding strand in one batch. The following (complementary) probes are suitable and preferable for this purpose:
SEQSEQTargetmethylated (5′->3′)IDunmethylated (5′->3′)IDAOX1TCGTTTCGGGTTTTCGAATTAGCG84TTGTTTTGGGTTTTTGAATTAGTG85(4 CpG) AOX1TTTTTGTTTTGGGTTTTTGAATTAGTG86(4 CpG) AOX1TCGTTTCGGGTTTTCGAATTAGCGC119TTGTTTTGGGTTTTTGAATTAGTGTG120(5 CpG)G AOX1TTTTTGTTTTGGGTTTTTGAATTAGTGTG121(5 CpG) RASSF1AGTGGTTTCGTTCGGTTCGCG122GTGGTTTTGTTTGGTTTGTG123(4 CpG) RASSF1ATTTGTGGTTTTGTTTGGTTTGTG124(4 CpG) RASSF1ATTTGTGGTTTTGTTTGGTTTGTGTTT125(4 CpG) TMAGAGGTTGTCGTTATCGGCGT87AGAGGTTGTTGTTATTGGTGT88(3 CpG) TMTTCGTATTCGTAGAGTCGTAGTCGG89TTTGTATTTGTAGAGTTGTAGTTGG90(4 CpG) TMAGAGGTTGTCGTTATCGGCGT165AGAGGTTGTCTTTATCTGCTT166(3 CpG) TMTTCGGCGTTTGTACGCGGCG167TTCTGCTTTTGTACTCTGCT168(5 CpG) TMGCGTTTGTACGCGGCGCGTT169GCTTTTGTACTCTGCTCTTT170(5 CpG) TMAGTTAGATTTGCGACGGATTGCG171AGTTAGATTTGCTACTGATTGCT172(3 CpG) TMCGGCGTTGGTCGTCGGCGTT173CTGCTTTGGTCTTCTGCTTT174(5 CpG) TMGCGTTGGTCGTCGGCGTTTT175GCTTTGGTCTTCTGCTTTTT176(4 CpG) TMGGCGTTGGTCGTCGGCGTTTT177GGCTTTGGTCTTCTGCTTTTT178(4 CpG) Septin-9ATTATGTCGGATTTCGCGGTTAACG179ATTATGTCTGATTTCTCTGTTAACT180(4 CpG) Septin-9TTATTATGTCGGATTTCGCGGTTAA181TTATTATGTCTGATTTCTCTGTTAACT182(4 CpG)C G Septin-9AGTTTGTGAGTGCGTGCGTTT183AGTTTGTGAGTGCTGCTTTT184(2 CpG)
As in the examples, the probes are particularly preferably 5′-FAM-marked (methylated DNA) or 5′-HEX-marked (unmethylated DNA) and marked with the quencher BHQ-1 at the 3′ end.
The object is also achieved by a kit for diagnosing a tumour disease in an isolated sample, containing:    i) primers for pre-amplifying methylated and unmethylated DNA sequences in the genes PLA2R1, RASSF1A and GSTP1 by means of PCR, each primer being selected such that forward and reverse primers together comprise up to seven, preferably from two to six, preferably from one to four, particularly preferably from one to three, more preferably from three to four, more preferably from four to five 5′-CG-3′ dinucleotide sequences,    ii) probes for quantifying the methylated and unmethylated DNA sequences in the genes PLA2R1, RASSF1A and GSTP1, each preferably comprising a fluorescent marker and a quencher as described above.
The primers used for the digital PCR are identical to those in the pre-amplification. Alternatively and preferably, the kit also contains the following:    iii) primers for the digital PCR for amplifying methylated and unmethylated DNA sequences in the genes PLA2R1, RASSF1A and GSTP1.
These additional primers are preferably used as intrinsic primers if extrinsic primers (acting as nested primers) were used in the pre-amplification.
The kit preferably contains additional components selected from the following:    iv) reaction buffers for the bias-based PCR amplification, preferably having a magnesium chloride concentration of from 0.5 to 15.0 mmol/l, preferably from 1.5 to 8 mmol/l, more preferably up to a final concentration of 3.5 mmol/l, particularly preferably of from 2.5 to 3.5 mmol/l, or alternatively a standard PCR buffer and a concentrated magnesium solution,    v) reaction buffers for the dPCR,    vi) deoxyribonucleotide mix,    vii) a DNA polymerase such as HotStarTaq Plus,    viii) control DNA, preferably an unmethylated DNA control and a methylated DNA control,    ix) instructions for use and optionally analysis software.
Preferably, the primers and probes and the control DNA for the kit are selected as above for the method. This applies in particular to the preferred targets of interest PLA2R1, RASSF1A and GSTP1, but also to the optional additional targets of interest AOX1, SERPINE1, thrombomodulin (TM) and/or septin-9.
DNA isolated from primary cells or cell lines is preferably used as control DNA for the method or kit according to the invention.
Preferably, DNA from cells or cell lines in which the target sequences are not methylated are used as the unmethylated control (negative control). Particularly preferably, DNA from epithelial cells from the healthy tissue corresponding to the tumour is used as the unmethylated DNA control. For example, DNA from human prostate epithelial cells (PrEC) or human mammary epithelial cells (HMEC) is preferably used as the unmethylated control for diagnosing prostate or breast cancer, respectively. Alternatively, DNA from MCF10A cell lines is used as the negative control for detecting breast cancer.
For each target of interest, DNA from a cell line in which the target sequence is homogeneously methylated is preferably selected as the methylated control (positive control) for detecting prostate cancer. DNA from the U937 leukaemia cell line and/or the LNCaP cell line is preferably used for PLA2R1, DNA from the U937 leukaemia cell line and/or the PC-3 cell line is preferably used for RASSF1A, and DNA from the U937 leukaemia cell line, the LNCaP cell line and/or the DU-145 cell line is preferably used for GSTP1. DNA from the DU-145 cell line is preferably used as the positive controls for SERPINE1 and thrombomodulin and/or DNA from the U937 leukaemia cell line and/or PC-3 cell line is preferably used for AOX1. DNA from the LNCaP, PC-3 and DU-145 cell lines is preferably used as the positive controls for septin-9.
To detect breast cancer, DNA from the MCF-7, Cal-51, UACC-812, BT-474, MDA-MB-453 and/or MDA-MB231 cell lines is preferably used as the positive controls.
Alternatively or in addition, DNA in which two out of three or three out of four 5′-CG-3′ dinucleotides are methylated is used as the positive control for heterogeneously methylated epialleles. DNA samples from the BPH-1 cell line in which epialleles have been detected and which are used as a comparison in the differential diagnosis of BPH, prostatitis and prostate cancer are particularly preferably used as controls for identifying and quantifying homogeneously and heterogeneously methylated epialleles. In particular for the genes PLA2R1, RASSF1A and/or GSTP1, DNA from the BPH-1 cell line is preferably used. When testing the benign prostate cell line BPH-1 in comparison with normal prostate epithelial cells (PrEC), the inventors were able to detect heterogeneously methylated DNA fragments, particularly in the targets of interest PLA2R1 and RASSF1A (FIGS. 18 and 19). When using probes having at least 3 CpG sites in their sequences, the heterogeneously methylated epialleles could be distinguished from the homogeneously methylated epialleles and quantified (FIG. 20-29). In this case, it was found that homogeneously methylated epialleles were specific to tumour DNA over heterogeneously methylated epialleles, and that their detection formed the basis for a clear-cut and thus more reliable differential diagnosis of benign and malignant diseases, in particular in the prostate. For this reason, probes containing at least three 5′-CG-3 or 5′-CA-3′ dinucleotides in their sequence are used in the dPCR following pre-amplification. Where these probes did not make it possible in individual cases to distinguish between e.g. benign hyperplasia such as BPH and malignant diseases such as prostate cancer, probes having four, five or more 5′-CG-3′ sites are used, or where this is not possible due to the gene sequences of the targets of interest or the probe design, a plurality of probes each having three 5′-CG-3′ or 5′-CA-3 dinucleotides for the same target of interest are used in the dPCR (step c)) of the method or kit according to the invention. In this way, a more clear-cut distinction is possible between benign and malignant diseases since the presence of a malignant tumour is only associated with the samples in which increased levels of homogeneously methylated epialleles are initially detected and quantified (i.e. four, five or six 5′-CG-3′ are methylated simultaneously). If this specification turns out to be too strict for detecting tumour DNA, resulting in the diagnostic sensitivity being too low, heterogeneously methylated epialleles having a correspondingly high number of methylated CpG sites (i.e. in each case at least two out of three 5′-CG-3′ dinucleotides methylated, or a total of four out of six tested 5′-CG-3′ dinucleotides simultaneously methylated) are preferably quantified and included in the data analysis. As already demonstrated by the inventors' results, heterogeneously methylated epialleles can be quantified when the aforementioned probe sequences are used (FIG. 20-29).
The invention also relates to the use of the kit for carrying out the method according to the invention.
Within the meaning of the invention, the term “tumour diagnosis” in particular includes early screening (precautionary examinations), prognosis, ongoing progress diagnosis, treatment monitoring and the detection of MRD.
In principle, the detection of circulating free tumour DNA using BBPA-dPCR can be used to diagnose any solid tumour, in particular when corresponding target gene sequences are present in bodily fluids or smears (liquid biopsies).
The method and kit according to the invention are particularly suitable for diagnosing malignant tumours such as cancers of the prostate, breast, renal cells, efferent urinary tract and bladder, pancreas, testicles, oral cavity and pharynx, gullet, larynx, thyroid, stomach, oesophagus, gut (in particular colorectal cancer), lungs, ovaries, cervix and uterus, gall bladder, malignant melanoma of the skin, astrocytoma, glioblastoma and neuroblastoma, as well as for diagnosing non-Hodgkin's lymphoma and Hodgkin's lymphoma, lymphoma of the skin, CNS, GI tract, stomach and intestinal lymphoma, and leukaemia.
In general, a preferred possible use of the method and kit is in the early detection (independent screening or precautionary examinations) of malignant diseases (including the diseases mentioned above), but in particular in combination with PSA determination and the indication for a tissue biopsy in the event of elevated PSA values and the diagnosis of prostate cancer, or in combination with mammograms and suspicious findings when diagnosing breast cancer, or in combination with the presence of a gene mutation entailing a higher family risk for breast or ovarian cancer, and the decision to opt for a prophylactic mastectomy and/or ovariectomy.
In one embodiment of the invention, screening or precautionary examinations are carried out on the basis of pooled samples, e.g. serum or plasma samples, by combining DNA sample material from e.g. 10 and 100 subjects/patients in a pool and first testing the 100-subject pool. If tumour DNA is identified in a pool of 100 subjects/patients, the samples from the 10-subject pool produced at the same time are analysed for the corresponding subjects/patient samples. If tumour DNA is detectable in one or more of these 10-subject pool samples, the individual samples are analysed. If no tumour DNA can be identified in the 100-subject pool samples, the 10-subject pool samples and individual samples are not tested. This means that a large number of samples can be screened for the presence of tumour DNA since the clear-cut amplification effect in the BBPA-dPCR method allows individual tumour DNA molecules to be detected, regardless of the concentration of background DNA.
If a tumour disease has been diagnosed, the progress of the disease can be monitored by identifying tumour DNA by means of BBPA-dPCR following surgery, chemotherapy or radiotherapy, and the presence of a minimal residual disease (MRD) can be diagnosed or ruled out. If no tumour DNA can be detected following treatment, this implies a good response to the treatment and an MRD can be ruled out. If tumour DNA can still be detected, this may indicate a need to optimise the treatment. If tumour DNA can be detected during the course of treatment when this was not the case previously, this implies a recurrence, which indicates the need to optimise the treatment again.
Advantageously, the method and kit according to the invention are particularly suitable for the differential diagnosis of benign diseases and malignant tumour diseases. The invention is most particularly suitable for the differential diagnosis of benign prostatic hyperplasia, prostatitis and prostate cancer, in particular when a prostate tissue biopsy has been indicated in line with the prior art due to elevated PSA values. Owing to the detection according to the invention of the methylation of PLA2R1, RASSF1A and GSTP1 (and optionally of AOX1, SERPINE1, thrombomodulin and/or septin-9) by means of the BBPA-dPCR, it is possible to determine the extent to which fcT-DNA can be detected in serum, plasma, urine and/or seminal fluid. On the basis of the determined methylation level, a distinction can be drawn between a benign prostatic hyperplasia, prostatitis and prostate cancer, and so unnecessary prostate tissue biopsies and operations can be prevented in many cases. If no tumour DNA can be found in e.g. a serum, plasma or urine sample by means of BBPA-dPCR, an additional PSA determination can be deferred (e.g. for three or six months). However, if there is tumour DNA detectable in the samples, a tissue biopsy is more strongly indicated.
Advantageously, the method and kit according to the invention are also particularly suitable for the differential diagnosis of breast cancer, in particular if a tissue biopsy is indicated on the basis of suspicious mammogram findings in line with the current prior art. Owing to the detection according to the invention of the methylation of PLA2R1, RASSF1A and GSTP1 (and optionally of AOX1, SERPINE1, thrombomodulin and/or septin-9) by means of the BBPA-dPCR, it is possible to determine the extent to which fcT-DNA can be detected in serum, plasma, urine and/or nipple secretions. On the basis of the determined methylation level, a distinction can be drawn between a benign microcalcification or cysts, and breast cancer, and so unnecessary tissue biopsies and operations can be prevented in many cases in the event of false-positive findings from the mammogram screening. Around two thirds of all women who begin annual mammograms from the age of 40 will be given false-positive results within the first ten years. An unnecessary biopsy is carried out in 7% of those cases [20]. Moreover, the method and corresponding kit according to the invention can reduce false-positive results in mammogram screenings.
The method and kit according to the invention are also suitable for the differential diagnosis of ovarian cancer, in particular when suspicious ultrasound findings indicate either a benign change, such as cysts, or ovarian cancer and further invasive diagnostic procedures according to the current prior art should be carried out.
In addition, in cases of a pathological gene mutation entailing a higher family risk for breast and ovarian cancer, e.g. in the BRCA1 and BRCA2 genes, the method and kit according to the invention are suitable as an additional decision-making tool as regards opting for a prophylactic mastectomy and/or ovariectomy, in particular if the patient in question is still planning a family.
The method according to the invention creates new possibilities in the diagnosis of tumour diseases, in particular in early diagnosis, since the method copies individual cf-tumour DNA copies in a suitably specific manner against a large background of normal wild-type DNA in the blood, urine or other biological samples before the subsequent quantification in the dPCR.
Even if the bias introduced leads to a change to the original ratio between methylated DNA fragments (as a sign of a malignant degeneration) and normal unmethylated wild-type DNA, the data determined by means of the BBPA-dPCR can be used for prognosis estimations (tumour load), the response to treatment (drop in fcT-DNA or constant fcT-DNA), detecting a minimal residual disease and tumour patient after-care (recurrence due to fcT-DNA increasing again). This is based on the fact that the degree of bias can be set variably in the method according to the invention by selecting the annealing temperature, magnesium concentration in the reaction buffer and the number of cycles, and that the bias is the same among the individual samples being tested when the conditions are constant, meaning that relative quantification is possible.
Another advantage of bias-inducing oligonucleotides (BIP) compared with methyl-specific oligonucleotides (MSP) is that the number of normal wild-type DNA fragments can be used as an internal control. In addition, using a primer pair that allows methylated and unmethylated DNA sequences to be simultaneously quantified prevents false results as a result of different portions of target of interest fragments and internal control gene fragments, such as ALU sequences.
The invention also relates to the primers according to sequences 1 to 19, 53, 61 to 76, 91 to 111, and 126 to 144, and to the nucleic acid probes according to sequences 20 to 52, 54 to 60, 77 to 90, 112 to 125, and 145 to 184, and to the use thereof for the aforementioned purposes.
The invention will be described in more detail on the basis of the following drawings and examples, without being limited thereto.